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Cell surface heparan sulfate proteoglycan syndecan-2 induces the maturation of dendritic spines in rat hippocampal neurons.

Ethell IM, Yamaguchi Y - J. Cell Biol. (1999)

Bottom Line: We demonstrate that the cell surface heparan sulfate proteoglycan syndecan-2 plays a critical role in spine development.Deletion of the COOH-terminal EFYA motif of syndecan-2, the binding site for PDZ domain proteins, abrogates the spine-promoting activity of syndecan-2.Our results indicate that syndecan-2 plays a direct role in the development of postsynaptic specialization through its interactions with PDZ domain proteins.

View Article: PubMed Central - PubMed

Affiliation: The Burnham Institute, La Jolla, California 92037, USA.

ABSTRACT
Dendritic spines are small protrusions that receive synapses, and changes in spine morphology are thought to be the structural basis for learning and memory. We demonstrate that the cell surface heparan sulfate proteoglycan syndecan-2 plays a critical role in spine development. Syndecan-2 is concentrated at the synapses, specifically on the dendritic spines of cultured hippocampal neurons, and its accumulation occurs concomitant with the morphological maturation of spines from long thin protrusions to stubby and headed shapes. Early introduction of syndecan-2 cDNA into immature hippocampal neurons, by transient transfection, accelerates spine formation from dendritic protrusions. Deletion of the COOH-terminal EFYA motif of syndecan-2, the binding site for PDZ domain proteins, abrogates the spine-promoting activity of syndecan-2. Syndecan-2 clustering on dendritic protrusions does not require the PDZ domain-binding motif, but another portion of the cytoplasmic domain which includes a protein kinase C phosphorylation site. Our results indicate that syndecan-2 plays a direct role in the development of postsynaptic specialization through its interactions with PDZ domain proteins.

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Postsynaptic localization of syndecan-2 on hippocampal neurons. (A–D) Double immunostaining of 30 DIV hippocampal neurons with antisynaptophysin (D, green) and anti–syndecan-2 (C, red) antibodies, and their overlap image (A and B).  (B) High-power magnification of the boxed area in A. Immunoreactivities of synaptophysin and syndecan-2 show close apposition with partial overlap (yellow) as seen in the synapsin I/heparan sulfate double staining (see Fig. 2, A–E). Bars, 20 μm in A–C  and 10 μm in D. (E–I) Double immunostaining with syndecan-2  (H, red) and PSD-95 (I, green) antibodies further confirmed synaptic localization of syndecan-2. (E–G) Punctate immunoreactivity for syndecan-2 was overlapping with PSD-95 immunolabeling  mostly along dendrite (see yellow), although occasionally there  were some PSD-95–positive dots (green) that are not positive for  syndecan-2 (arrows in G), that may represent PSD-95 immunoreactivity at nonsynaptic sites on dendritic shaft (see Results). Bars,  20 μm in E and 7 μm in F.
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Figure 5: Postsynaptic localization of syndecan-2 on hippocampal neurons. (A–D) Double immunostaining of 30 DIV hippocampal neurons with antisynaptophysin (D, green) and anti–syndecan-2 (C, red) antibodies, and their overlap image (A and B). (B) High-power magnification of the boxed area in A. Immunoreactivities of synaptophysin and syndecan-2 show close apposition with partial overlap (yellow) as seen in the synapsin I/heparan sulfate double staining (see Fig. 2, A–E). Bars, 20 μm in A–C and 10 μm in D. (E–I) Double immunostaining with syndecan-2 (H, red) and PSD-95 (I, green) antibodies further confirmed synaptic localization of syndecan-2. (E–G) Punctate immunoreactivity for syndecan-2 was overlapping with PSD-95 immunolabeling mostly along dendrite (see yellow), although occasionally there were some PSD-95–positive dots (green) that are not positive for syndecan-2 (arrows in G), that may represent PSD-95 immunoreactivity at nonsynaptic sites on dendritic shaft (see Results). Bars, 20 μm in E and 7 μm in F.

Mentions: To determine which syndecan is responsible for the spine-specific accumulation of heparan sulfate, hippocampal neurons at 30 DIV were stained with pAbs to syndecan-2 and syndecan-4 (Kim et al., 1994). This analysis revealed that syndecan-4 is not expressed in neurons, but in astrocytes which were present as a minor population in older cultures (data not shown). On the other hand, syndecan-2 is expressed in neurons in a time course and a pattern similar to those of heparan sulfate immunoreactivity. Syndecan-2 was localized on the surface of dendrites and cell bodies of hippocampal pyramidal neurons in a punctate pattern (Fig. 4, D–F). Syndecan-2 immunoreactivity was first detected at 2 wk in vitro. Then punctate staining became even stronger at 3 and 4 wk in vitro (Fig. 4, E and F). Double staining for synaptophysin, a presynaptic marker, and syndecan-2 showed partially overlapping patterns of staining, similar to the result of synapsin I/heparan sulfate double staining (see Fig. 2, A–C). Double staining with anti–syndecan-2 and anti–PSD-95 antibodies further confirmed a synaptic localization of syndecan-2 (Fig. 5, E–I). Punctate immunoreactivity for syndecan-2 and PSD-95 showed significant overlap mostly along dendrites (Fig. 5, E–G), although occasionally there were some PSD-95– positive dots that were not positive for syndecan-2 (see arrows in Fig. 5 G). These puncta are likely to represent PSD-95 at nonsynaptic sites on dendritic shafts (Aoki, C., Z. Shusterman, M. Kasat, M. Bak, L. Alexandre, and D.S. Bredt. 1998. Society for Neuroscience Annual Meeting. Abstract 713.11). Together, these results strongly suggest that syndecan-2 is the cell surface HSPG predominantly localized on the dendritic spines of cultured hippocampal neurons. In adult rat brain, syndecan-2 has been shown recently to be highly concentrated at asymmetric synapses formed on the dendritic spines of pyramidal neurons in the CA3 area of the hippocampus (Hsueh et al., 1998). These observations further support our finding that syndecan-2 is one of the HSPGs responsible for the spine-specific accumulation of heparan sulfate on mature hippocampal neurons in vitro, though a contribution by unknown HSPGs has not been ruled out.


Cell surface heparan sulfate proteoglycan syndecan-2 induces the maturation of dendritic spines in rat hippocampal neurons.

Ethell IM, Yamaguchi Y - J. Cell Biol. (1999)

Postsynaptic localization of syndecan-2 on hippocampal neurons. (A–D) Double immunostaining of 30 DIV hippocampal neurons with antisynaptophysin (D, green) and anti–syndecan-2 (C, red) antibodies, and their overlap image (A and B).  (B) High-power magnification of the boxed area in A. Immunoreactivities of synaptophysin and syndecan-2 show close apposition with partial overlap (yellow) as seen in the synapsin I/heparan sulfate double staining (see Fig. 2, A–E). Bars, 20 μm in A–C  and 10 μm in D. (E–I) Double immunostaining with syndecan-2  (H, red) and PSD-95 (I, green) antibodies further confirmed synaptic localization of syndecan-2. (E–G) Punctate immunoreactivity for syndecan-2 was overlapping with PSD-95 immunolabeling  mostly along dendrite (see yellow), although occasionally there  were some PSD-95–positive dots (green) that are not positive for  syndecan-2 (arrows in G), that may represent PSD-95 immunoreactivity at nonsynaptic sites on dendritic shaft (see Results). Bars,  20 μm in E and 7 μm in F.
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Related In: Results  -  Collection

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Figure 5: Postsynaptic localization of syndecan-2 on hippocampal neurons. (A–D) Double immunostaining of 30 DIV hippocampal neurons with antisynaptophysin (D, green) and anti–syndecan-2 (C, red) antibodies, and their overlap image (A and B). (B) High-power magnification of the boxed area in A. Immunoreactivities of synaptophysin and syndecan-2 show close apposition with partial overlap (yellow) as seen in the synapsin I/heparan sulfate double staining (see Fig. 2, A–E). Bars, 20 μm in A–C and 10 μm in D. (E–I) Double immunostaining with syndecan-2 (H, red) and PSD-95 (I, green) antibodies further confirmed synaptic localization of syndecan-2. (E–G) Punctate immunoreactivity for syndecan-2 was overlapping with PSD-95 immunolabeling mostly along dendrite (see yellow), although occasionally there were some PSD-95–positive dots (green) that are not positive for syndecan-2 (arrows in G), that may represent PSD-95 immunoreactivity at nonsynaptic sites on dendritic shaft (see Results). Bars, 20 μm in E and 7 μm in F.
Mentions: To determine which syndecan is responsible for the spine-specific accumulation of heparan sulfate, hippocampal neurons at 30 DIV were stained with pAbs to syndecan-2 and syndecan-4 (Kim et al., 1994). This analysis revealed that syndecan-4 is not expressed in neurons, but in astrocytes which were present as a minor population in older cultures (data not shown). On the other hand, syndecan-2 is expressed in neurons in a time course and a pattern similar to those of heparan sulfate immunoreactivity. Syndecan-2 was localized on the surface of dendrites and cell bodies of hippocampal pyramidal neurons in a punctate pattern (Fig. 4, D–F). Syndecan-2 immunoreactivity was first detected at 2 wk in vitro. Then punctate staining became even stronger at 3 and 4 wk in vitro (Fig. 4, E and F). Double staining for synaptophysin, a presynaptic marker, and syndecan-2 showed partially overlapping patterns of staining, similar to the result of synapsin I/heparan sulfate double staining (see Fig. 2, A–C). Double staining with anti–syndecan-2 and anti–PSD-95 antibodies further confirmed a synaptic localization of syndecan-2 (Fig. 5, E–I). Punctate immunoreactivity for syndecan-2 and PSD-95 showed significant overlap mostly along dendrites (Fig. 5, E–G), although occasionally there were some PSD-95– positive dots that were not positive for syndecan-2 (see arrows in Fig. 5 G). These puncta are likely to represent PSD-95 at nonsynaptic sites on dendritic shafts (Aoki, C., Z. Shusterman, M. Kasat, M. Bak, L. Alexandre, and D.S. Bredt. 1998. Society for Neuroscience Annual Meeting. Abstract 713.11). Together, these results strongly suggest that syndecan-2 is the cell surface HSPG predominantly localized on the dendritic spines of cultured hippocampal neurons. In adult rat brain, syndecan-2 has been shown recently to be highly concentrated at asymmetric synapses formed on the dendritic spines of pyramidal neurons in the CA3 area of the hippocampus (Hsueh et al., 1998). These observations further support our finding that syndecan-2 is one of the HSPGs responsible for the spine-specific accumulation of heparan sulfate on mature hippocampal neurons in vitro, though a contribution by unknown HSPGs has not been ruled out.

Bottom Line: We demonstrate that the cell surface heparan sulfate proteoglycan syndecan-2 plays a critical role in spine development.Deletion of the COOH-terminal EFYA motif of syndecan-2, the binding site for PDZ domain proteins, abrogates the spine-promoting activity of syndecan-2.Our results indicate that syndecan-2 plays a direct role in the development of postsynaptic specialization through its interactions with PDZ domain proteins.

View Article: PubMed Central - PubMed

Affiliation: The Burnham Institute, La Jolla, California 92037, USA.

ABSTRACT
Dendritic spines are small protrusions that receive synapses, and changes in spine morphology are thought to be the structural basis for learning and memory. We demonstrate that the cell surface heparan sulfate proteoglycan syndecan-2 plays a critical role in spine development. Syndecan-2 is concentrated at the synapses, specifically on the dendritic spines of cultured hippocampal neurons, and its accumulation occurs concomitant with the morphological maturation of spines from long thin protrusions to stubby and headed shapes. Early introduction of syndecan-2 cDNA into immature hippocampal neurons, by transient transfection, accelerates spine formation from dendritic protrusions. Deletion of the COOH-terminal EFYA motif of syndecan-2, the binding site for PDZ domain proteins, abrogates the spine-promoting activity of syndecan-2. Syndecan-2 clustering on dendritic protrusions does not require the PDZ domain-binding motif, but another portion of the cytoplasmic domain which includes a protein kinase C phosphorylation site. Our results indicate that syndecan-2 plays a direct role in the development of postsynaptic specialization through its interactions with PDZ domain proteins.

Show MeSH
Related in: MedlinePlus